Lipoprotein Assay

Abstract

The present invention concerns a method of determining the concentration of total lipoprotein in a sample. The method involves the steps of: (i) adding to an aliquot of the sample a lipophilic dye that binds to lipoproteins in the sample and which when so bound fluoresces under appropriate excitation; and (ii) determining the total lipoprotein concentration in the sample using fluorescence analysis. A method of analysing the lipoprotein content of a sample solution using a dye that discriminates between different types of lipoprotein is also disclosed.

1 . A method of determining the concentration of total lipoprotein in a sample, the method comprising the steps of:—(i) adding to an aliquot of the sample a lipophilic dye that binds to lipoproteins in the sample and which when so bound fluoresces under appropriate excitation; and (ii) determining the total lipoprotein concentration in the sample using fluorescence analysis. 
     
     
         2 . The method of  claim 1 , wherein the lipophilic dye has two phenol groups that may be substituted or unsubstituted. 
     
     
         3 . The method of  claim 2 , wherein the lipophilic dye is a chalcone dye. 
     
     
         4 . The method of  claim 3 , wherein the chalcone dye is 4-methylaminomethylchalcone or K-37. 
     
     
         5 . The method of  claim 2 , wherein the lipophilic dye is a dye comprising the fluorescent unit: Ph-[C—C═C] n —C-Ph 
     
     
         6 . The method of  claim 5 , wherein the dyes is diphenylhexatriene or diphenyloctatetrene. 
     
     
         7 . The method of  claim 1 , wherein the dye is a Coumarin dye. 
     
     
         8 . The method of  claim 7 , wherein the dye is Coumarin 30. 
     
     
         9 . The method of  claim 1 , wherein the method further comprises adding to the aliquot a ligand binding inhibitor that substantially inhibits the binding of the dye to a hydrophobic binding domain on Human Serum Albumin before lipoprotein concentrations are determined. 
     
     
         10 . The method of  claim 9 , wherein the ligand binding inhibitor comprises a fatty acid or a functional derivative thereof. 
     
     
         11 . The method of  claim 10 , wherein the ligand binding inhibitor comprises octanoic acid (Cg) or a derivative thereof. 
     
     
         12 . A method of analysing the lipoprotein content of a sample solution, the method comprising the steps of:—
 (a) adding to a first aliquot of the sample a lipophilic dye that binds to lipoproteins in the sample and which when so bound fluoresces under appropriate excitation; 
 (b) determining the total lipoprotein concentration in the first aliquot using fluorescence analysis; 
 (c) adding to a second aliquot of the sample a discriminating dye that binds to a specific lipoprotein or lipoproteins in the sample and which when so bound fluoresces under appropriate excitation; 
 (d) determining the concentration of the lipoproteins in the second aliquot using fluorescence analysis; and 
 (e) calculating the lipoprotein content by comparing the concentrations determined in steps (b) and (d). 
 
     
     
         13 . The method of  claim 12 , wherein the specific lipoprotein is HDL. 
     
     
         14 . The method of  claim 13 , wherein the discriminating dye is Nile Red. 
     
     
         15 . The method of  claim 14 , wherein the concentration of Nile Red added to the sample is between approximately 0.1-0.9 mM. 
     
     
         16 . The method of  claim 12 , wherein the lipophilic dye is a dye selected from the group consisting of a calcone dye and a Coumarin dye. 
     
     
         17 . The method of  claim 12 , wherein the method further comprises adding to the second aliquot a ligand binding inhibitor that substantially inhibits the binding of the dye to a hydrophobic binding domain on Human Serum Albumin before lipoprotein concentrations are determined. 
     
     
         18 . The method of  claim 12 , wherein the sample is a biological fluid. 
     
     
         19 . The method of  claim 12 , wherein the sample comprises blood plasma or serum, or lymph. 
     
     
         20 . The method of  claim 12 , wherein the lipophilic dye is a dye selected from the group consisting of 4-methylaminomethylchalcone, K-37, diphenylhexatriene, diphenyloctatetrene, and Coumarin 30. 
     
     
         21 . The method of  claim 17 , wherein the ligand binding inhibitor comprises a fatty acid or a functional derivative thereof. 
     
     
         22 . The method of  claim 21 , wherein the ligand binding inhibitor comprises octanoic acid (Cg) or a derivative thereof. 
     
     
         23 . The method of  claim 1 , wherein the sample is a biological fluid. 
     
     
         24 . The method of  claim 1 , wherein the sample comprises blood plasma or serum, or lymph.